Accepted Manuscript Polyethyleneimine anchored copper(II) complexes: Synthesis, characterization, In vitro DNA binding studies and cytotoxicity studies Jagadeesan Lakshmipraba, Sankaralingam Arunachalam, Anvarbatcha Riyasdeen, Rajakumar Dhivya, Mohammad Abdulkader Akbarsha PII: DOI: Reference:

S1011-1344(14)00351-0 http://dx.doi.org/10.1016/j.jphotobiol.2014.11.005 JPB 9876

To appear in:

Journal of Photochemistry and Photobiology B: Biology

Received Date: Revised Date: Accepted Date:

18 August 2014 15 November 2014 19 November 2014

Please cite this article as: J. Lakshmipraba, S. Arunachalam, A. Riyasdeen, R. Dhivya, M.A. Akbarsha, Polyethyleneimine anchored copper(II) complexes: Synthesis, characterization, In vitro DNA binding studies and cytotoxicity studies, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/ j.jphotobiol.2014.11.005

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Polyethyleneimine anchored copper(II) complexes: Synthesis, characterization, In vitro DNA binding studies and cytotoxicity studies Jagadeesan Lakshmipraba,a Sankaralingam Arunachalam,*,a Anvarbatcha Riyasdeen,b Rajakumar Dhivya,c Mohammad Abdulkader Akbarsha,b a

School of Chemistry, Bharathidasan University, Tiruchirappalli - 620 024, Tamil Nadu, India b

Mahatma Gandi-Doerenkamp Centre, Bharathidasan University, Tiruchirappalli - 620 024, Tamil Nadu, India

c

Department of Biomedical Science, Bharathidasan University, Tiruchirappalli - 620 024, Tamil Nadu, India

Abstract The water soluble polyethyleneimine-copper(II) complexes, [Cu(phen)(L-tyr)BPEI]ClO4 (where phen = 1,10-phenanthroline, L-tyr

= L-tyrosine and

BPEI =

branched

polyethyleneimine) with various degree of copper(II) complex units in the polymer chain were synthesized and characterized by elemental analysis and electronic, FT-IR, EPR spectroscopic techniques. The binding of these complexes with CT-DNA was studied using UV-visible absorption titration, thermal denaturation, emission, circular dichroism spectroscopy and cyclic voltammetric methods. The changes observed in the physicochemcial properties indicated that the binding between the polymer–copper complexes and DNA was mostly through electrostatic mode of binding. Among these complexes, the polymer-copper(II) complex with the highest degrees of copper(II) complex units (higher degrees of coordination) showed higher binding constant than those with lower copper(II) complex units (lower degrees of coordination) complexes. The complex with the highest number of metal centre bound strongly due to the cooperative binding effect. Therefore, anticancer study was carried out using this complex. The cytotoxic activity for this complex on MCF-7 breast cancer cell line was determined adopting MTT assay, acridine orange/ethidium bromide (AO/EB) staining and comet assay techniques, which revealed that the cells were committed to specific mode of cell death either apoptosis or necrosis. Keywords: polyethyleneimine-copper(II) complex / DNA interaction / cytotoxic studies fax: +91-431-2407043, Email address: [email protected]

1. Introduction During the past decade, there has been tremendous interest in the synthesis and studies pertaining to the interaction of various metal complexes with DNA and also screening of these complexes for their anticancer activities so as to replace cisplatin [1-5]. Hence, much attention has been targeted on the design of metal-based complexes, which can bind to DNA. Interaction of metal complexes with DNA should be useful in the development of molecular probes and new therapeutic agents. Cancer research mainly targets the DNA molecule which is the origin of uncontrolled cell division. To block this cell division, there is a need for developing DNA targeted chemotherapy drugs. Several small molecules/metal complexes have been interacted with DNA to correlate their effects of binding/cleavage behavior on cytotoxicity [6-7]. In these aspects of drug designing, some of the issues like solubility, transfection efficiency, targeted delivery, etc. may enter as practical problems. To overcome these problems, drug carriers like cationic polymers, surfactants, liposomes and dentrimers were employed for efficient drug delivery [8]. Copper is a physiologically important metal element that plays an important role in the endogenous oxidative DNA damage associated with aging and cancer [9]. Copper(II) complexes bearing 1,10-phenanthroline ligand have been widely used due to their high nucleolytic efficiency [10] and numerous biological activities such as antitumor, anti-candida and antimicrobial activities [11,12]. It has been reported that a binuclear copper(II) complex containing 1,10-phenanthroline and a trinuclear copper(II) complex containing di-(2picolyl)amine bind strongly with DNA and cleave more effectively than their corresponding monomeric complexes [13-15]. The reports in the literature that advocate design studies on metal complexes that cooperative effect arising from such non-covalent interactions would be a

valuable principle in the development of new metal based probes which recognize biomolecular targets with high specificity [16]. Recent literature indicate that mixed ligand copper(II) complexes have been receiving considerable attention for various reasons. Copper(II) complexes having amino acid ligand are of interest due to their bioloigcal relavance, good DNA binding ability, antimicrobial and anticancer activities [17]. Also, there are reports on drug polymer conjugates as potential candidates for the selective delivery of anticancer agents to tumor tissues. Particularly, polyethyleneimine (PEI), possesses quite a number of advantages as polymer chelating agent, such as good water solubility, high content of functional groups, suitable molecular weights as well as good physical and chemical stabilities [18]. Stable polyethyleneimine-copper(II) complexes are reported where copper ions are hard to elute from the polymer domain to the bulk solution and binding constant values are 1 to 5 order magnitude greater in the polymer-chelate systems than in the monomeric Cu-complex systems [19]. Our research group has been involved in the synthesis and studies on mixed polymercopper(II) complexes and their DNA binding properties for the past several years [20,21]. We have reported synthesis and nucleic acid binding of polyethyleneimine-copper(II) complexes with L-valine and L-arginine as a co-ligand [22,23]. In the present work we report the synthesis, DNA binding and antitumor properties of polyethyleneimine-copper(II) complexes containing Ltyrosine as co-ligand. L-tyrosine contains benzene ring which can influence the binding of these complexes with DNA through π-π interactions.

2. Experimental section 2.1. Materials

Calf thymus DNA and branched polyethyleneimine (BPEI) (Mw ca. 25,000) were obtained from Sigma-Aldrich, Germany and were used as obtained. Copper(II) chloride dihydrate, 1,10-phenanthroline (Merck, India) and tyrosine (Loba Chemie, India) and were used as received. The precusor complex, [Cu(phen)(L-tyr)(H2O)]ClO4, was prepared as reported earlier [24]. A solution of calf thymus DNA in the buffer gave a UV absorbance ratio of ~1.8-1.9 : 1 at 260 and 280 nm, indicating that the DNA was sufficiently free of protein. The concentration of CT DNA in base pairs was determined by UV absorbance at 260 nm by taking the molar extinction coefficient value 13200 M-1 cm-1 for DNA at 260 nm [25,26]. All the experiments involving the interaction of the polymer-copper(II) complex with DNA were carried out using buffer containing 5 mM Tris–HCl/50 mM NaCl at pH 7.0 in twice distilled water. MCF-7 cell line was obtained from National Centre For Cell Science (NCCS), Pune. 2.2. [Cu(phen)(L-tyr)BPEI]ClO4 To a solution of branched polyethyleneimine (BPEI) (0.15 g, 3.4 mmol) dissolved in ethanol (15 ml), [Cu(phen)(L-tyr)(H2O)]ClO4 (0.8 g, 1.4 mmol) in water was added slowly with stirring. The mixture was heated between 50-60 ºC for 15 h in a water bath with stirring. The resulting dark blue solution was dialyzed at 15 ºC against distilled water for 4-5 days. The solvent was then evaporated in a rotary evaporator under reduced pressure at room temperature. The dark-bluish filmy substance obtained was pulverized and dried. Yield = 0.23 g for x = 0.182. (Anal. Calc.: C, 33.77, H, 4.40, N, 14.50, O, 5.81, Cu 18.35, Found: H, 5.02, N, 14.25, O, 5.77, Cu 18.37% (x = 0.182 obtained from carbon content). IR (KBr, cm-1): ν(N-H) 3446, ν(C-C) 2924, ν(COO-) 1099, ν(C=C) 1471, ν(C=N)1390, 1099, ν(C-H) 852, ν(C-H) 730; UV (λmax, nm,(ε, M-1 cm-1)): 227 (28,120) 272(54,240), 294(41,830), 645(11,090). EPR (77 K, g║ 2.211 and g┴ 2.017; RT giso = 2.0753.

Polymer-copper(II) complex samples with various numbers of copper(II) complex units bound to the polymer chain were synthesized by changing the amount of reactants in the reaction solution, the duration of the reaction time and the reaction temperature. 2.3. Physical measurements Elemental analysis was determined at Sophisticated Analytical Instrument Facility (SAIF), Lucknow, India. Absorption spectra and thermal denaturation studies were recorded on a UV-Vis-NIR Cary300 Spectrophotometer using cuvettes of 1 cm path length in tris buffer solution, and emission spectra were recorded on a JASCO FP 770 spectrofluorimeter. FT-IR spectra were recorded on a FT-IR JASCO 460 PLUS spectrophotometer with samples prepared as KBr pellets. EPR spectra were recorded on a JEOL-FA200 EPR spectrometer at room temperature and at LNT in methanol solution. Absorption titration experiments of polymer– copper(II) complexes in buffer (50 mM NaCl–5 mM Tris–HCl, pH 7.0) were performed by using a fixed complex concentration to which increments of the DNA stock solutions were added. Polymer-copper(II) complex-DNA solutions were incubated for 10 min before the absorption spectra were recorded. Equal amount of DNA was added to both the complex and reference solutions to eliminate the absorbance of DNA itself. For fluorescence quenching experiments DNA was pretreated with ethidium bromide (EB) for 30 min. The polymer–copper(II) complexes were then added to this mixture and their effect on the emission intensity was measured. Samples were excited at 450 nm and emission was observed between 500 and 700 nm. Circular dichroism spectra were recorded at room temperature using the same tris buffer. For the cyclic voltammetry experiments, the electrode surfaces were freshly polished with alumina powder and then sonicated in ethanol and distilled water for 1 min prior to each experiment. Then the electrodes were rinsed throughly with

distilled water. Cyclic voltammetric experiments were performed at 25.0 ± 0.2 ºC in a single compartment cell with a three-electrode configuration (glassy carbon working electrode, platinum wire auxiliary electrode and saturated calomel reference electrode). The solution was deoxygenated with nitrogen gas for 20 min prior to experiments. 2.4. Cell culture The MCF-7 cancer cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (Sigma, USA) and 10000 IU of penicillin and 100 µg ml-1 of streptomycin as antibiotics (Himedia, Mumbai, India), in 96 well culture plates, at 37 oC, in a humidified atmosphere of 5% CO2, in a CO2 incubator (Forma, Thermo Scientific, USA). All the experiments were performed using cells from passage 15 or less. 2.4.1. Cytotoxicity assay (MTT assay) The polymer-copper(II) complex of the highest degree of coordination was dissolved in DMSO, diluted in culture medium and used to treat the model cell line over a complex concentration range of 3 to 30 µg ml-1 for a period of 24 h and 48 h. DMSO at 0.5% concentration in the culture medium was used as negative control. We have used cisplatin as positive control. A miniaturized viability assay using 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl2H-tetrazolium bromide (MTT) (Sigma, USA) (5 mg/ml in Phosphate-Buffered Saline (PBS)) was added to each well and the plates were wrapped with aluminium foil and incubated at 37 ˚C for 4 h. By this treatment a purple formazone product was formed due to the reduction of MTT by the mitochondrial enzyme succinate dehydrogenase of the cells [27]. The purple formazan product was dissolved by addition of 100 µl of 100% DMSO to each well. The absorbance was monitored at 570 nm (measurement) and 630 nm (reference) using a 96 well plate reader (Bio-

Rad, Hercules, CA, USA). Data were collected for four replicates each and used to calculate the respective means. The percentage of inhibition was calculated, from this data, using the formula: [Mean absorbance of untreated cells (control) - Mean absorbance of treated cells (test)] x 100. Mean absorbance of untreated cells (control)

The IC50 value was determined as the complex drug concentration that is required to reduce the absorbance to half that of the control. 2.4.2. Acridine orange (AO) and ethidium bromide (EB) staining Acridine orange/ethidium bromide staining was performed as follows: the cell suspension of each sample containing 5 × 105 cells, was treated with 25 µl of AO and EB solution (1 part of 100 µg ml-1 AO and 1 part of 100 µg ml-1 EB in PBS) and examined in a fluorescent microscope (Carl Zeiss, Germany) using an UV filter (450-490 nm). Three hundred cells per sample were counted in tetraplicate for each dose point. The cells were scored as viable, apoptotic or necrotic as judged by the staining, nuclear morphology and membrane integrity [28], and the percentages of apoptotic and necrotic cells were then calculated. Morphological changes were also observed and photographed. 2.4.3. Comet assay DNA damage was detected by adopting comet assay as reported earlier [29]. Cells were suspended in low-melting-point agarose in PBS and pipetted out to microscope slides pre-coated with a layer of normal-melting-point agarose. Slides were chilled on ice for 10 min and then immersed in lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 0.2 mM NaOH, pH 10.01 and Triton X-100) and the solution was kept for 4 h at 4 oC in order to lyse the cells and to permit DNA unfolding. Thereafter, the slides were exposed to alkaline buffer (300 mM NaOH, 1

mM Na2EDTA, pH > 13) for 20 min to allow DNA unwinding. The slides were washed with buffer (0.4 M Tris, pH 7.5) to neutralize excess alkali and to remove detergents before staining with ethidium bromide (5 µl in 10 mg ml-1). Photographs were obtained using the fluorescent microscope. One hundred and fifty cells from each treatment group were digitalized and analyzed using CASP software. The images were used to estimate the DNA content of individual nuclei and to evaluate the degree of DNA damage representing the fraction of total DNA in the tail.

4. Results and discussion 4.1. Degree of coordination The structure of the water soluble polymer-copper(II) complex is shown in Fig. 1. In this figure ‘x’ represents the degree of coordination, which is the number of moles of copper(II) chelate per mole of the repeating unit (amine group) of polymeric ligand. If the entire repeating units (amine group) in the polymer are coordinated to copper, then the value of x is 1. The degree of coordination (x) was calculated from carbon content value [19,30,31] . The degree of coordination thus obtained for the polymer-copper(II) complex samples of the present work are 0.059, 0.149, 0.182. The stability of the polymer–copper(II) complexes in solution was verified occasionally by keeping the solution of the polymer–copper(II) complexes in dialysis bags, small amount of the solution from the dialysis bags was taken and the stability was confirmed through absorption spectroscopy. Any free copper complex ion or copper ion in the solution outside the dialysis bag was not observed (which was verified using spectrophotometric method), indicating that polymer-complexes were very stable during the handling of our experiments. Also viscosity, is a significant experiment which gives information about interaction in aqueous solution. In our

case, even after dialysis we do not have change in viscosity. This proves that the complexes are very stable in solution. 4.2. Characterization of polymer-copper(II) complexes The FT-IR spectra for the polymer-copper(II) complexes displayed stretching frequencies around 1471 cm-1 and 1390 cm-1 which can be attributed to the ring stretching frequencies viz., ν(C=C) and ν(C=N), respectively, of the coordinated 1,10-phenanthroline and these are at slightly lower freuqencies than that of uncoordinated 1,10-phenanthroline. The ν(C-H) out-ofplane bending values, around 852 cm-1 and 730 cm-1, for the phenanthroline ligand were shifted to 838 cm-1 and 693 cm-1, respectively, in the complexes. These shifts can be explained by the fact that each of the two nitrogen atoms of phenanthroline ligands donates a pair of electrons to the central copper atom forming coordinate bond [32]. The band obtained around 2924 cm-1 can be assigned to C-C stretching vibration of aliphatic CH2 of BPEI whereas the broad band observed around 3446 cm-1 can be assigned to the N-H stretching of BPEI [33]. The uncoordinated amino acid exhibited a stretch in the region 1750-1700 cm-1 corresponding to ν(COOH). In the complexes, this band was shifted to 1630 cm-1 indicating the coordination of carboxylate group to the copper(II) ion. The very strong band around 1099 cm-1 can be assigned to the presence of perchlorate anion. The stretching frequencies around 508 cm-1 and 491 cm-1 can be attributed, respectively, to copper-nitrogen and copper-oxygen stretching. The UV-visible absorption spectra of all the complexes were recorded in the region 200800 nm. All the complexes displayed four bands in the regions 230-645 nm. In the UV region, the absorption bands below 300 nm are attributed to intra-ligand transitions whereas in the visible region, the band around 645 nm is assigned as d-d transition.

The solid state EPR spectra of the polymer-copper(II) complex (x = 0.203) was recorded in X-band frequencies at room temperature as well as in frozen solution (77 K) in methanol as solvent (Fig. 2). The room temperature EPR spectra of the polymer-copper(II) complexes showed single isotropic feature at giso = 2.070-2.075, and this broadening of isotropic peak is due to intermolecular spin exchange. This intermolecular type of spin exchange is caused by the strong spin coupling which occurs during a coupling of two paramagnetic species. At liquid nitrogen temperatue we observed three peaks with the third being broad. This is because the copper complex units have been mounted on a polymer chain resulting in some spin–spin coupling between the copper complex units which leads to a small amount of broadening. In liquid nitrogen temperature the complexes showed the values of g║ = 2.211-2.216 and g┴ = 2.016-2.020. The existence of g║ > g┴ > 2.00 suggests that dx2-dy2 is the ground state with the d9 (Cu2+) configuration and square pyramidal geometry. 4.3. Absorption studies Electronic absorption spectroscopy is an effective method to examine the binding mode of DNA with polymer-copper(II) metal complexes. Thus, in order to provide evidence for the binding of polymer-copper(II) complexes to DNA, the binding process was monitored by absorption spectroscopy by following the changes in absorption band intensity and its position. On addition of DNA, the absorption spectra of polymer-copper(II) complex showed hyperchromism and slight red shift (Fig. 3). The experimental results derived from the UVvisible titration experiments suggest that positively charged polymer-complexes can bind to DNA, probably to the phosphate groups, by electrostatic interaction resulting in the stabilization of DNA duplex. Nevertheless, the metal complex units present in the polymer chain contain aromatic moieties so the binding of the complexes involving partial intercalation of an aromatic

ring between the base pairs of DNA cannot be ruled out. From the above studies the intrinsic binding constants (Kb) were determined from the increase of absorption at 294 nm calculated by absorption spectral titration. In order to compare quantitatively the binding affinity with nucleic acids between polymer-copper(II) complexes having different degrees of coordination, the intrinsic binding constants Kbs of the complexes were determined using equation (4) by assuming a simple model, in which the reaction between the nucleic acid site, P and the copper complex unit of the polymer complex, D to form the nucleic acid bound complex, PD as: P+D

PD

----- (1)

Kb = [PD]/[P][D] ----- (2) where, [PD], [P] and [D] represent the respective equilibrium concentrations of nucleic acid bound copper complex units, nucleic acid sites in base pairs and the copper complex units of the polymer complex. A = εD[D]+ εPD[PD] ----- (3) CD/A-εDCD= (1/εPD – εD ) + 1/(εPD – εD )Kb 1/[P] -----(4) where, εD and εPD are the molar extinction coefficient of the free copper complex units and apparent molar extinction coefficient of the nucleic acid bound copper complex units respectively, CD total concentration of copper complex units and A is the experimental absorbance. An iterative procedure was employed as per the method provided in the reference [34] to arrive at the Kb values (first [D] set equal to CD, then, once a first estimate of Kb and (εPDεD) are obtained, a new value of [D] was calculated and so on until convergence is achieved). This procedure yields a binding constant value (Kb) for each complex As seen from the Table 1 the binding constants observed for polymer-copper(II) complexes are higher those that of similar type of simple metal complexes like [Cu(phen)(L-tyr)

H2O]ClO4 (Kb = 3.75 x 103 M-1) as well as the polymer alone PEI (Kb = 1.2 M-1) [35,36]. However, they are very much lower than the potential intercalators like ethidium bromide (Kb, 7.0 x 107 M-1 in 40 mM Tris/HCl, pH 7.9) [37] and the partially intercalating complexes like [Co(phen)2(dppz)]3+ (Kb = 9.09 x 105 M-1) and [Ru(imp)2(dppz)]2+ (Kb = 2.19 x 107 M-1) [38], which implies that these complexes bind to DNA relatively less strongly than classical intercalators and partial intercalators. Also, as seen from the Table, it was observed that the binding constant changes with degree of coordination of copper(II) units in the polymer chain; greater the ratio of copper(II) centres in the polymer chain, higher was the binding constant because when one copper(II) complex unit binds with DNA it will cooperatively act to increase the overall binding ability of the other copper(II) complex units to DNA. 4.4. Ethidium bromide displacement assay All polymer–copper(II) complexes were non-emissive upon excitation of the MLCT band, either in aqueous solution or in the presence of DNA. The competitive binding experiments with a well-established quenching assay based on the displacement of the intercalating EB from ct-DNA was carried out in order to get further information regarding the DNA binding properties of polymer-metal complexes. The quenching of emission intensity of DNA bound EB (Fig. 4) was analyzed through Stern-Volmer equation, I0/I = 1 + Ksv[Q], where I0 and I are the fluorescence intensities in the absence and presence of the complex, respectively, Ksv is the linear Stern-Volmer constant and Q is the concentration of polymer-copper(II) complex [39,40]. A plot of I0/I vs. [Q] was drawn and Ksv was obtained from the ratio of slope to intercept (Table 1). As seen from the Table, the Ksv value increases with increase in degree of coordination of polymer-copper(II) complex. This is attributed to the cooperative binding

between copper(II) units on the same polymer chain with DNA. This cooperative effect increases with degree of coordination. 4.5. Effect of ionic strength The change in fluorescence intensity of cationic copper(II)-polymer complexes to DNA in the presence of NaCl can be used to verify whether the binding mode is electrostatic or intercalative; a linear relation between fluorescence intensity and concentration of NaCl is highly indicative of an electrostatic mode of interaction whereas a non-dependence of fluorescence intensity on ionic strength indicates intercalation [41,42]. It is observed that as the concentration of NaCl increases, the relative fluorescence intensity due to ethidium bromide increases (Fig. 5). This is due to the competitive binding of Na+ ions to DNA which decreases the binding affinity of the copper(II)-polymer complex to DNA. As the concentration of NaCl increases, a linear increase of fluorescence is noticed, indicating that the cationic copper(II)-polymer complexDNA interactions for the polymers studied are electrostatic. [43,44] 4.6. Circular dichroism spectral studies CD spectral technique is useful method to monitor the conformational variations of DNA during complex-DNA interactions and achieve information on changing DNA conformation by the binding of the metal complex to DNA. DNA has a major longwave positive peak centred at 275 nm and the intensity of this positive peak is similar in magnitude to that of the negative peak centred at 245 nm (Fig. 6) corresponding to the π-π stacking of the base pairs and right handed helicity of B-form DNA in buffer solution [45]. Addition of polymer-copper(II) complex (x = 0.182) to B-form DNA has been shown to induce a B to A transition, resulting in a CD spectrum with characteristics totally different from those of B-form DNA; the long wave positive peak is larger with a maximum at ~270 nm and a very large shortwave peak results below ~230 nm [46].

Thus, the increased ellipticity observed at 275 nm when polymer-copper(II) complex binds to DNA can be interpreted as unwinding of B form of DNA due to a decrease in twist angle. This can be tentatively interpreted as B form of DNA becoming more ‘A-like’ upon binding polymercopper(II) complex. 4.7. Cyclic voltammetry studies The binding of polymer-copper(II) complexes with DNA was further confirmed by cyclic voltammetric studies. The cyclic voltammogram of polymer-copper(II) complex (x = 0.182) in the absence and presence of DNA is shown in Fig. 7. In the absence of DNA, the cathodic peak potential (Epc) and the anodic peak potential (Epa) of our complex are 339 mV and 574 mV, respectively, with a large peak-to-peak separation, ∆Ep, of 235 mV and the ratio of cathodic to anodic peak current (ipc/ipa) is 1.05 indicating a quasi-reversible redox process [47]. The formal potential (E1/2) which is taken as the average of Epc and Epa is 0.457 V in the absence of DNA, whereas in the presence of DNA a negative shift in E1/2 by 0.055V along with increase in ∆Ep of 30 mV has been observed. The ipc/ipa value also increased with the increase of the DNA concentration. Literature report [48] have pointed out that the shift direction of electrochemical potential of metal complex, after reacting with DNA, is related to its binding mode with DNA. A positive shift of the peak potential and the negative shift indicating that electrostatic mode of interaction. The dependence of cathodic current on scan rate was also investigated. In the case of our polymer-copper(II) complex the plot of cathodic current vs. the square root of the scan rate (ν1/2), was linear for the complex alone, and the complex in the presence of DNA, which indicates that the electrochemical process are diffusion controlled process [48]. 5. Cytotoxic assay

5.1. MTT assay In vitro cytotoxicity of polymer-copper(II) complexes was evaluated by MTT assay on MCF-7 cells. The cytotoxic effects of the polymer-copper(II) complex of the highest degree of coordination was examined on cultured MCF-7 human breast cancer cells by exposing cells for 24 h and 48 h to medium containing the complex at 3-30 mg ml-1 concentration (Fig. 8). The polymer-copper(II) complex inhibited the growth of the cancer cells significantly, in a dose- and duration-dependent manner. The cytotoxic activity was determined according to the dose values of the exposure of the complex required to reduce survival to 50% (IC50), compared to untreated cells. The IC50 values of the complexes are 20.4 ± 2.5 and 14.3 ± 1.7 µg ml-1 after 24 h and 48 h respectively. The polymer-copper (II) complex showed highly effective cytotoxic activity against MCF-7 cancer cells and the IC50 value of the complex was lesser for 48 h treatment group than for 24 h treatment group. Inspite of its high cytotoxic activity against MCF-7 cells, the cytotoxic effectiveness was relatively lower when compared to cisplatin, the IC50 values of which were 13.71± 0.5 and 12.56± 0.8 µg ml-1 for 24 h and 48h treatment periods, respectively. However, cytotoxic potential apart, cisplatin has been established to produce toxic side effects [49] which is not expected with the polymer-copper(II) complex in present study [50]. The cytotoxic effect of the polymer-copper(II) complex may be interpretable as due to its amphiphilic nature [51] and, hence, would penetrate the cell membrane easily, reduce the energy status in tumors and also to alter hypoxia status in the cancer cell microenvironment, which are factors that would influence the antitumor acidity. It is known that phenanthroline-containing metal complexes have a wide range of biological activities such as antitumor, antifungal, apoptosis [52-54], interaction with DNA thereby inhibiting replication, transcription, and other nuclear functions and arresting cancer cell proliferation so as to arrest tumor growth.

5.2. Assessment of cell death based on morphological features Apoptosis is a gene-controlled cell death process, which is characterized by DNA fragmentation, chromatin condensation and marginalization, membrane blebbing, cell shrinkage, and fragmentation of cells into membrane-enclosed vesicles or apoptotic bodies to be phagocytosed by macrophages [55]. To further confirm the mode of cell death induced by the complex on cancer cells

AO/EB (Acridine Orange/Ethidium Bromide) staining (Apoptosis

Assays) was adopted, which would reveal the changes in the gross cytology of the cell with special reference to cytoplasm and nucleus. After treatment of MCF-7 cancer cells, polymercopper(II) complex of the highest degree of coordination, at the respective IC50 concentrations for 24 h and 48 h, the cells were observed for the gross cytological changes. The treated cells revealed all the above cytological changes (Fig. 9). These cytological changes indicated that the cells were committed to cell death, mostly, apoptosis and to a certain extent necrosis. 5.3. Single-cell gel electrophoresis (Comet assay) Among the different techniques used for measuring and analyzing DNA strand breaks in mammalian cells, the single cell gel electrophoresis assay (Comet assay) is considered as a rapid, simple, visual and sensitive technique to asses DNA fragmentation typical of toxic DNA damage and of an early stage of apoptosis [54]. As shown in Fig. 10, the images were used to estimate the DNA content of individual nuclei and to evaluate the degree of DNA damage representing the fraction of total DNA in the tail. Cells were assigned to five groups: 0-20% (intact), 20-40% (slightly damaged), 40-60% (damaged), 60-80% (highly damaged) and >80% (dead). The results revealed that DNA damage was induced in MCF-7 cancer cells by the polymer-copper(II) complex, and the incidence was greater at 48 h than at 24 h, as shown in Fig. 10.

Conclusions

Water

soluble

polyethyleneimine

coordinated-copper(II)

complexes

containing

phenanthroline and L-tyrosine as co-ligands with various degrees of coordination were synthesised. The complexes were characterized adopting various spectroscopic techniques and elemental analysis. The binding between the polymer-copper(II) complexes and DNA was assessed in relation to the polymer complex with different degrees of copper complex content in the polymer chain. The electronic absorption spectral studies, emission studies and ionic strength effect showed that these complexes bind to DNA via electrostatic modes of binding. These studies indicates that the binding affinity towards DNA increases with the increase in the number of copper centres in the polymer. The changes in circular dichroism and cyclic voltammetry studies of the binding between one of our complexes in the presence of DNA confirm the above mentioned modes of binding. Thermal denaturation studies of the binding between our complexes and DNA reveal that the complex with higher degree of coordiantion binds with DNA and stability enhanced. The polymer-copper(II) complex of the highest degree of coordination showed good cytotoxic activity against MCF-7 cancer cell with mostly through apoptosis although a few cells succumbed to necrosis

Acknowledgments We are grateful to the UGC-SAP and DST-FIST programmes of the Department of Chemistry, Bharathidasan University. Council of Scientific and Industrial Research (CSIR), New Delhi is acknowledged for financial support [Scheme. No. 09/475(0154)/2010-EMR-I dated. 09/02/2011] for Senior Research Fellowship to JLP. One of the authors, SA., thanks for sanction of research schemes, Grant No. SR/S1/IC-13/2009 of DST, Grant No. 01(2461)/11/EMR-II of CSIR and also Grant No. 41-223/2012(SR) of UGC. Grants from Doerenkamp-Zbinden

Foundation, Switzerland, and King Saud University, Riyadh, Kingdom of Saudi Arabia to MAA are gratefully acknowledged. References [1] [2]

[3]

[4] [5] [6] [7]

[8] [9] [10] [11]

[12]

[13]

[14]

[15] [16]

J.B. Chaires, Drug-DNA interactions, Curr. Opin. Chem. Biol. 8 (1998) 314–320. K.J. Du, J.Q. Wan, J.F. Kou, G.Y. Li, L.L. Wang, H. Chao, L.N. Ji, Synthesis, DNAbinding and topoisomerase inhibitory activity of ruthenium(II) polypyridyl complexes, Eur. J. Med. Chem. (2011) 1056–1065. S. Roy, K.D. Hagen, U. Maheswari, M. Lutz, A.L. Spek, J. Reedijk, G.P. van Wezel, Phenanthroline Derivatives with Improved Selectivity as DNA-Targeting Anticancer or Antimicrobial Drugs, Chem. Med. Chem. 3 (2008) 1427–1434. P. Borst, S. Rottenberg, J. Jonkers, How do real tumors become resistant to cisplatin?, Cell Cycle 15 (2008) 1353–1359. X. Wang, Fresh platinum complexes with promising antitumor activity, Anticancer Agents Med. Chem. 10 (2010) 396–411. W.H. Ang, P.J. Dyson, Classical and non-classical ruthenium-based anticancer drugs: Towards targeted chemotherapy, Eur. J. Inorg. Chem. 2006 (2006) 4003–4018. X.-L. Zhao, Z.-S. Li, Z.-B. Zheng, A.-G. Zhang, K.-Z. Wang, pH luminescence switch, DNA binding and photocleavage, and cytotoxicity of a dinuclear ruthenium complex, Dalton Trans. 42 (2013) 5764–5777. R. Chadha, V.K. Kapoor, D. Thakur, R. Kaur, P. Arora, D.V.S. Jain, Drug carrier systems for anticancer agents: A review, J. Sci. Ind. Res. 67 (2008) 185–197. B.N. Ames, M.K. Shigenaga, T.M. Hagen, Oxidants, antioxidants, and the degenerative diseases of aging, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 7915–7922. D.S. Sigman, Chemical nucleases, Biochemistry 29 (1990) 9097–1105. G. Majella, S. Vivienne, M. Malachy, D. Michael, M.Vickie, Synthesis and anti-Candida activity of copper(II) and manganese(II) carboxylate complexes: X-ray crystal structures of [Cu(sal)(bipy)]·C2H5OH·H2O and [Cu(norb)(phen)2]·6.5H2O (salH2=salicylic acid; norbH2=cis-5-norbornene-endo-2,3-dicarboxylic acid; bipy=2,2-bipyridine; phen=1,10phenanthroline), Polyhedron 18 (1999) 2931–2939. J.D. Saha, U. Sandbhor, K. Shirisha, S. Padhye, D. Deobagkar, C.E. Ansond, A.K. Powell, A novel mixed-ligand antimycobacterial dimeric copper complex of ciprofloxacin and phenanthroline, Bioorg. Med. Chem. Lett. 14 (2004) 3027–3032. S. Zhang, Y. Zhu, C. Tu, H. Wei, Z. Yang, L. Lin, J. Ding, J. Zhang, Z. Guo, A novel cytotoxic ternary copper(II) complex of 1,10-phenanthroline and l-threonine with DNA nuclease activity, J. Inorg. Biochem. 98 (2004) 2099–2106. Y. Ni, D. Lin, S. Kokot, Synchronous fluorescence, UV–visible spectrophotometric, and voltammetric studies of the competitive interaction of bis(1,10-phenanthroline)copper(II) complex and neutral red with DNA, Anal. Biochem. 352 (2006) 231–242. T. Gupta, S. Dhar, M. Nethaji, A.R. Chakravarty, Bis(dipyridophenazine)copper(II) complex as major groove directing synthetic hydrolase, Dalton Trans. (2004) 1896–1900. A.D. Burrows, C.W. Chan, M.M. Chowdhry, J.E. McGrady, D.M.P. Mingos, Multidimensional crystal engineering of bifunctional metal complexes containing complementary triple hydrogen bonds, Chem. Soc. Rev. 24 (1995) 329–339.

[17] M. Chauhan, K. Banerjee, F. Arjmand, DNA Binding Studies of Novel Copper(II) Complexes Containing L-Tryptophan as Chiral Auxiliary: In Vitro Antitumor Activity of Cu-Sn2 Complex in Human Neuroblastoma Cells, Inorg. Chem. 46 (2007) 3072–3082. [18] B.L. Rivas, K.E. Geckeler, Synthesis and metal complexation of poly(ethylenimine) and derivatives, Adv. Polym. Sci. 102 (1992) 171–188. [19] E. Tsuchida, H. Nishide, T. Ohkawa, Bulletin of the Science and Engineering Research Laboratory, Waseda University, 69 (1975) 31–34. [20] R.S. Kumar, K. Sasikala, S. Arunachalam, DNA interaction of some polymer–copper(II) complexes containing 2,2′-bipyridyl ligand and their antimicrobial activities, J. Inorg. Biochem. 102 (2008) 234–241. [21] J. Lakshmipraba, S. Arunachalam, Studies on the interactions of polymer-anchored copper(II) complexes with tRNA, Transition Met. Chem. 35 (2010) 477–482. [22] J. Lakshmipraba, S. Arunachalam, D.A. Gandi, T. Thirunalasundari, Synthesis, nucleic acid binding and cytotoxicity of polyethyleneimine-copper(II) complexes containing 1,10phenanthroline and L-valine, Eur. J. Med. Chem. 46 (2011) 3013–3021. [23] J. Lakshmipraba, S. Arunachalam, A. Riyasdeen, R. Dhivya, S. Vignesh, M.A. Akbarsha, R.A. James, DNA/RNA binding and anticancer/ antimicrobial activities of polymer– copper(II) complexes, Spectrochim. Acta, Part A 109 (2013) 23–31. [24] T. Sugimori, H. Masuda, N. Ohata, K. Koiwai, A. Odani, O. Yamauchi, Structural Dependence of Aromatic Ring Stacking and Related Weak Interactions in Ternary Amino Acid-Copper(II) Complexes and Its Biological Implication, Inorg. Chem. 36 (1997) 576– 583. [25] T. Lundback, T. Hard, Sequence-specific DNA-binding dominated by dehydration, Proc. Natl. Acad. Sci. 93 (1996) 4754–4759. [26] R. Marty, C.N. N’soukpoé-Kossi, D.M. Charbonneau, L. Kreplak, H.-A. Tajmir-Riahi, Structural characterization of cationic lipid-tRNA complexes, Nucleic Acids Res. (2009) 1–11. [27] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods. 65 (1983) 55–63. [28] D.L. Spector, R.D. Goldman, L.A. Leinwand, Cell: A Laboratory Manual, vol. 1, Culture and Biochemical Analysis of Cells, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1998) pp. 34.1–34.9. [29] N.P. Singh, M.T. MacCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell 175 (1988) 184–191. [30] Y. Kurimura, E. Tsuchida, M. Kaneko, Preparation and Properties of Some Water Soluble Co(III)-poly-4-vinylpyridine Complexes, J. Polym. Sci., Part A-1 9 (1971) 3511–3519. [31] J.W. Kolis, D.E. Hamilton, N.K. Kildahl, Nickel(II) and copper(II) complexes of linear pentadentate ligands derived from substituted o-hydroxyacetoand ohydroxybenzophenones, Inorg. Chem. 18 (1979) 1826–1831. [32] A.A. Schilt, R.C. Taylor, Infrared spectra of 1,10-phenanthroline metal complexes in the rock salt region below 2000 cm−1, J. Inorg. Nucl. Chem. 9 (1959) 211–221. [33] K.E. Geckeler, G. Lange, H. Eberhardt, E. Bayer, Preparation and application of watersoluble polymer-metal complexes, Pure Appl. Chem. 52 (1980) 1883–1905. [34] T. Biver, B. García, J. M. Leal, F. Secco and E. Turriani, Left-handed DNA: intercalation of the cyanine thiazole orange and structural changes. A kinetic and thermodynamic approach, Phys. Chem. Chem. Phys. 12(2010) 13309–13317.

[35] Q.-Q. Zhang, F. Zhang, W.-G. Wang, X.-L. Wang, Synthesis, crystal structure and DNA binding studies of a binuclear copper(II) complex with phenanthroline, J. Inorg. Biochem. 100 (2006) 1344–1352. [36] Y.-I. Zhou, Y.-Z. Li. The interaction of poly(ethylenimine) with nucleic acids and its use in determination of nucleic acids based on light scattering, Spectrochim. Acta, Part A 60 (2004) 377–384. [37] M.J. Waring, Complex formation between ethidium bromide and nucleic acids, J. Mol. Biol. 13 (1965) 269–282. [38] J.G. Liu, B.H. Ye, H. Li, L.N. Ji, R.H. Li, J.Y. Zhou, Synthesis, characterization and DNAbinding properties of novel dipyridophenazine complex of ruthenium (II) : [Ru(IP)2(DPPZ)]2+, J. Inorg. Biochem. 73 (1999) 117–122. [39] S. Ramakrishnan, M. Palaniandavar, Interaction of rac-[Cu(diimine)3]2+ and rac[Zn(diimine)3]2+ complexes with CT DNA: effect of fluxional Cu(II) geometry on DNA binding, ligand-promoted exciton coupling and prominent DNA cleavage, Dalton Trans. (2008) 3866–3878. [40] N. Shahabadi, S. Kashanian, M. Khosravi, M. Mahdavi, Multispectroscopic DNA interaction studies of a water-soluble nickel(II) complex containing different dinitrogen aromatic ligands, Transition Met. Chem. 35 (2010) 699–705. [41] S. Kashanian, M. M. Khodaei, H. Roshanfekr, N. Shahabadi, G. Mansouri, DNA binding, DNA cleavage and cytotoxicity studies of a new water soluble copper(II) complex: The effect of ligand shape on the mode of binding Spectrochim. Acta - Part A 86 (2012) 351– 359. [42] S. Kashanian, M. M. Khodaei, H. Roshanfekr, H. Peyman, DNA interaction of [Cu(dmp)(phen-dion)] (dmp = 4,7 and 2,9 dimethyl phenanthroline, phen-dion = 1,10phenanthroline-5,6-dion) complexes and DNA-based electrochemical biosensor using chitosan-carbon nanotubes composite film, Spectrochim. Acta - Part A 114 (2013) 642649. [43] M.T. Jr Record, T.M. Lohman, P. de Haseth, Ion effects on ligand– nucleic acid interactions,. J. Mol. Biol. 107 (1976) 145–158. [44] M.X. Tang, F. C. Szoka, The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes, Gene Therapy 4 (1997) 823–832. [45] K. van Holde, W.C. Johnson, P.S. Ho, Principles of Physical Biochemistry; (1998) Prentice Hall: New York. [46] S. Zhang, Y. Zhu, C. Tu, H. Wei, Z. Yang, L. Lin, J. Ding, J. Zhang, Z. Guo, A novel cytotoxic ternary copper(II) complex of 1,10-phenanthroline and L-threonine with DNA nuclease activity, J. Inorg. Biochem. 98 (2004) 2099–2106. [47] T. Carter, M.A. Rodriguez, A.J. Bard, Voltammetric Studies of the Interaction of Metal Chelates with DNA. 2. Tris-Chelated Complexes of Cobalt(III) and Iron(II) with 1,10Phenanthroline and 2,2'-Bipyridine, J. Am. Chem. Soc. 111 (1989) 8901–8911. [48] M.T. Carter, A.J. Bard, Voltammetric studies of the interaction of tris(1,10phenanthroline)cobalt (III) with DNA, J. Am. Chem. Soc. 109 (1987) 7528–7530. [49] A.-M. Florea, D. Büsselberg, Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects, Cancers 3 (2011) 1351–1371. [50] G.J. Brewer, Copper control as an antiangiogenic anticancer therapy: lessons from treating Wilson's disease, Exp. Biol. Med. (Maywood) 226 (2001) 665–673.

[51] J.P. Behr, Gene transfer with synthetic cationic amphiphiles. Prospects for gene therapy, Bioconjug. Chem. 5 (1994) 382–389. [52] B. Coyle, P. Kinsella, M. McCann, M. Devereux, R. O’Connor, M. Clynes, K. Kavanagh, Induction of apoptosis in yeast and mammalian cells by exposure to 1,10-phenanthroline metal complexes, Toxicol. In Vitro 18 (2004) 63–70. [53] B. Coyle, M. McCann, K. Kavanagh, M. Devereux, M. Geraghty, Mode of anti-fungal activity of 1,10-phenanthroline and its Cu(II), Mn(II) and Ag(I) complexes, BioMetals 16 (2003) 321–329. [54] M. McCann, M. Geraghty, M. Devereux, D. O’Shea, J. Mason, L. O’Sullivan, Insights into the mode of action of the anti-Candida activity of 1,10-phenanthroline and its metal chelates, Metal-Based Drugs 7 (2000) 185–193. [55] P. Moller, L.E. Knudsen, S. Loft, H. Wallin, The comet assay as a rapid screen test in biomonitoring occupational exposure and effect of confounding factors, Cancer Epidemiol. Biomarkers Prevent. 9 (2000) 1005–1015. [54] M.A. Maire, C. Rast, Y. Landkocz, P. Vasseur, 2,4-Dichlorophenoxyacetic acid: effects on Syrian hamster embryo (SHE) cell transformation, c-Myc expression, DNA damage and apoptosis, Mutat. Res. 631 (2007) 124–136.

Figure captions Fig.1. Schematic representation of [Cu(phen)(L-tyr)BPEI]ClO4 Fig. 2. EPR spectrum of [Cu(phen)(L-tyr)(BPEI)]ClO4 (x = 0.203) in methanol at liquid nitrogen temperature (inset: solid state EPR spectrum at room temperature). Fig. 3. Absorption spectra of [Cu(phen)(L-tyr)BPEI]ClO4 (x = 0.182) in the absence of DNA and in the presence of DNA, [complex] = 3 x 10-5 M, [DNA] = 0-3.2 x 10-5 M (inset: plot of [DNA]/(εa-εf) vs. [DNA]) Fig. 4. Emission spectra of EB bound to DNA, [EB] = 2 x 10-4 M in the absence of complex and in the presence of complex (x = 0.182), [DNA] = 2 x 10-3 M, [complex] = 0-1 x 10-4 M Fig. 5. Titration of DNA[DNA] = 2 x 10-4 M in the presence of ethidium bromide[EB] = 2 x 10-4 M in the presence of polymer-copper(II) complexes[complex] = 1 x 10-4 M as a function of NaCl concentration. Fig. 6. Circular dichroism spectra in the absence (black) and in the presence [Cu(phen)(Ltyr)BPEI]ClO4 (x = 0.182), [complex] = 12 x 10-5 M (red) with DNA, [DNA] = 9 x 10-5 M Fig. 7. Cyclic voltammograms of [Cu(phen)(L-tyr)BPEI]ClO4 (x = 0.182), [complex] = 1 x 10-3 M (black) in the presence of DNA (red) [DNA] = 0 – 8.0 x 10-4 M, scan rate: 50 mV s-1 Fig. 8. Inhibition of in vitro cancer cells growth by [Cu(phen)(L-tyr) BPEI]ClO4 (x = 0.182). Fig. 9. Photomicrographs of control (the cells were viable as inferred from the green fluorescence) and AO/EB stained MCF-7 cancer cells treated with the [Cu(phen)(Ltyr)BPEI]ClO4 (x = 0.182) at 20.4 and 14.3 µg ml-1 concentration for 24 and 48 h. Scale bar: 35µ m. The graph shows data on percentage of cells that are normal afflicted with apoptosis and necrosis in the control and 24h and 48h treatment groups. Fig. 10. Comet images of DNA double strand breaks at 12 and 24 h treatment of [Cu(phen)(Ltyr)BPEI]ClO4 (x = 0.182) at 20.4 and 14.3 µg ml-1 concentration. Cells were grown in RPMI1640 medium containing FBS at 10% final concentration, and streptomycin (10 mg ml-1) and penicillin (10,000 IU ml-1) as antibiotics. The duration-dependence of the DNA damage is revealed. Scale bar: 35µm. DNA damage in MCF-7 cell populations as defined according to the percentage of DNA in the tail.

N H O

N H (1-x)

O

Cu N N NH2 .ClO4

x

OH

Fig. 1.

Fig. 2.

1.0

0.0000030

[DNA]/ε a−ε f

Absorbance

0.0000025

0.0000020

0.0000015

0.0000010

0.0000005

0.5

0.00001

0.00002

0.00003

[DNA]

0.0 200

300

400

Wavelength, nm

Fig. 3.

500

600

400

Intensity

300

200

100

0 500

600

Wavelength, nm

Fig. 4.

700

1.0

Relative Fluoresence

0.8

0.6

0.4

1 2 3

0.2

0.0 0.0

0.2

0.4

[NaCl] mM

Fig. 5.

0.6

0.8

80

DNA DNA+ complex

a

CD, m deg

40

0

-40

-80 240

280

Wavelength, nm

Fig. 6.

320

Fig. 7.

Fig. 8.

100

Normal Apoptosis Necrosis

80

% of cells

60

40

20

0 Control

24h

Fig. 9.

48h

140

Dead Highly Damaged Damaged Slightly Damaged Intact

120

% of cells

100

80

60

40

20

0 Control

24 h

Fig. 10.

48 h

Table 1 The intrinsic binding constant (Kb) of [Cu(phen)(L-tyr)BPEI]ClO4, with DNA and RNA and thermal melting temperature in the presence of [Cu(phen)(L-tyr)BPEI]ClO4 with different degree of coordination. Complex

Degree of

Kb (M-1)

Ksv (M-1)

coordination (x)

± 0.04

± 0.03

0.059

2.10 x 104

2.13 x 104

0.149

2.03 x 105

2.78 x 104

0.182

7.80 x 105

3.37 x 104

[Cu(phen)(L-tyr)BPEI]ClO4

• • •

Polymer-copper(II) complexes with different ‘x’ containing L-tyrosine as co-ligand synthesized DNA binding interaction was done for the complexes These complexes bind to DNA via electrostatic modes